Abstract/summary Proteases work in crowded intracellular environments occupied by thousands of potential protein substrates. Thus, knowing how these destructive enzymes choose the right proteins for degradation is critical for understanding both their biological functions and those of specific substrates. Intracellular proteases play important roles in eliminating damaged or harmful proteins, in resculpting the proteome following changes in gene expression, in cell-cycle control, and as sensor and regulatory components in stress-response pathways. Our primary goal is to determine the molecular mechanisms that allow peptide signals in substrates, adaptors, and other regulatory molecules to interact with proteases to control protein degradation in bacteria. A second practical goal is to develop synthetic systems of targeted degradation that test basic principles and provide community tools for studying protein function. ATP-dependent degradation of cytoplasmic proteins by AAA+ proteases occurs in all organisms. Biochemical, genetic, and structural studies will elucidate fundamental molecular mechanisms of substrate recognition for three Escherichia coli AAA+ proteases (ClpXP, HslUV, and Lon), and provide paradigms for understanding how orthologs of these enzymes identify the correct intracellular substrates in other bacteria and eukaryotes. Regulated intramembrane proteolysis (RIP) is a method of signal transduction. We will determine the detailed molecular mechanisms that allow a PDZ-protease (DegS) to sense envelope stress in the periplasm of E. coli and initiate a proteolytic cascade that relays information across the inner membrane to a second PDZ-protease (RseP). The molecular logic of this signaling system and its control by input signals and interactions of the PDZ- proteases with two regulatory proteins (RseA and RseB) will be elucidated. We will also dissect a related RIP system (AlgW-MucA-MucB) that controls alginate biosynthesis in Pseudomonas aeruginosa. Understanding intracellular degradation is a key goal of basic research, with applications in biotechnology and medicine. For example, knowing how substrates are identified will enable improved bacterial expression of recombinant proteins, and controlled degradation systems will permit validation of new antibiotic targets. AAA+ proteases often play roles in the virulence of bacterial pathogens and thus can be antibiotic targets. Moreover, mutations in the MucA and MucB proteins of the P. aeruginosa RIP system increase mortality and morbidity in patients with cystic fibrosis. Finally, understanding DegS function will be relevant to studies of its human homolog, HtrA2/Omi, which contributes to caspase- independent apoptosis and cancer prevention. Relevance Understanding intracellular degradation is a key goal of basic research, with applications in biotechnology and medicine. For example, knowing how substrates are identified would allow improved bacterial expression of recombinant proteins, and controlled-degradation systems would permit validation of novel antibiotic targets. AAA+ proteases often play roles in the virulence of bacterial pathogens and thus can be antibiotic targets. Moreover, mutations in the MucA and MucB proteins of the P. aeruginosa RIP system increase mortality and morbidity in patients with cystic fibrosis. Finally, understanding DegS function will be relevant to studies of its human homolog, HtrA2/Omi, which contributes to caspase-independent apoptosis and cancer prevention.